Enzyme immunoassays (EIA) have become important analytical methods in clinical chemistry laboratories for the selective detection of drugs, hormones, and proteins at trace levels. As specific examples, such methods are now used routinely to detect diagnostically important blood proteins, including creatine kinase-MB to detect the occurrence of myocardial infarctions, prostate-specific antigen (PSA) to screen for prostate cancer, and human chorionic gonadotropin (hCG) to confirm pregnancy. Further, such methods can be used to detect the protein coat of a specific virus. Thus, the invention also has utility for assays in agriculture and the food processing industry, for example, in-line monitoring of a food processing plant.
One of the must useful of the immunoassays is the two-antibody sandwich technique, which is used primarily to determine the presence and concentration of an antigenic analyte in an unknown sample. Two-antibody assays can be relatively quick, particularly if a source of pure antigen is available. The assay requires two antibodies that bind to non-overlapping epitopes on the antigenic analyte. Either two monoclonal antibodies that recognize discrete sites or one batch of affinity-purified polyclonal antibodies can be used.
To use the conventional two-antibody assay, a "capture" antibody is purified and bound to a solid phase, and the antigenic analyte in a standard or test sample solution is allowed to bind to the capture antibody. Unbound analyte proteins are then typically removed by washing. A second antibody, which is labeled with an enzyme, is allowed to bind to the immuno-bound analyte. After another wash step to remove the excess enzyme-labeled antibody, the amount of enzyme label bound to the matrix is determined, usually by adding a substrate on which the enzyme acts and measuring the rate of product generated. The rate that product is generated is directly proportional to the amount of the antigenic analyte present in the sample.
This sandwich-type assay is superior to other types of solid phase immunoassays with respect to sensitivity, specificity and kinetics. These advantages arise from the fact that excess amounts of the capture antibody and the enzyme-antibody conjugate are used relative to the low levels of the analyte commonly present, thus driving the equilibrium of the binding reaction toward the formation of the sandwich structure.
An EIA method can be heterogeneous or homogeneous, depending on whether or not washing steps are required to separate free and bound enzyme label. EIA's can also be competitive or non-competitive, depending on the availability of antibody binding sites. Of these different EIA methods, non-competitive heterogeneous sandwich assays have the advantage of using two co-existing determinant sites on the same antigenic analyte to be detected. As a result, these sandwich-type assays generally exhibit good specificity when identifying the presence and/or amount of protein analytes. Further, due to the innate amplification properties of enzymes, the use of EIA offers excellent sensitivity.
Even though sandwich-type EIA's are used widely in clinical laboratories, such methods usually require multiple wash steps to separate the excess amounts of reagents used from the sandwich being formed. This has created a need for complex instrumentation to do a high volume of immunoassay tests. Thus, the need to limit the wash steps has made it very difficult to adapt these assays into portable test systems that would be desirable for detecting diagnostically important proteins in field locations, such as doctors' offices, emergency vehicles, and hospital "crash" carts.
Another problem has been the length of time required for incubation to accomplish sufficient binding between wash steps. While immunoconcentration techniques using capture antibodies immobilized on glass and other filter material have reduced the binding incubation times usually associated with sandwich-type EIA's, discrete separation and wash steps are still required. For example, the use of suspensions of particles possessing immobilized antibodies can greatly enhance the speed of immmobinding. However, then, additional filtering or centrifugation steps are required.
Further, such immunoconcentration methods are only useful for detecting proteins found in the circulatory system when the red blood cells have been removed. Serum and plasma samples are required, rather than whole blood, because the presence of red blood cells clogs the filter-based devices used in immunoconcentration techniques. See Valkirs et al., "Qualitative Two-site IEMA for Serum hCG Using Immunoconcentration Technology", Clin. Chem. 31, 960 (1985) and Valkirs et al., "Immunoconcentration--a New Format for Solid-Phase Immunoassays", Clin. Chem. 31, 1427-31 (1985).
The development of a rapid, simple, non-separation method for the detection of proteins has been a long-standing goal. Gibbons et al., "Homogeneous Enzyme Immunoassay for Proteins Employing .beta.-Galactosidase", Anal. Biochem. 102, 167-170 (1980), and Armenta et al., "Improved Sensitivity in Homogeneous Enzyme Immunoassays Using a Fluorogenic Macromolecular Substrate: An Assay for Serum Ferritin", Anal. Biochem. 146, 211-219 (1985), used chromogenic and fluorogenic galactoside-dextran substrates in devising homogeneous enzyme immunoassays for C-reactive protein, ferritin, and immunoglobulins. However, the low degree of modulating enzyme activity in this homogeneous protocol has rendered the method impractical for real world applications.
Chen et al., "Ultrasound-Accelerated Immunoassay, as Exemplified by Enzyme Immunoassay of Choriogonadotropin", Clin. Chem. 30, 1446-51 (1984), combined a two-enzyme channeling technique with immunocapillary migration to produce a test-strip format for the detection of hCG. Although wash steps were not required, this approach is not truly separation-free, since the test trip has to be removed from the sample before adding the enzyme substrate.
Schray et al., "Separation-Free Dual Solid Phase Enzyme Immunoassay for Macromolecules", Anal. Chem., 60 353-56 (1988), have reported a separation-free dual solid-phase enzyme immunoassay for macromolecules, which relies on the partitioning of an enzyme conjugate (biotin-glucose-6-phosphate dehydrogenase-antibody) between two solid phases of polystyrene latex-bound avidin and polystyrene latex-bound analyte. However, this assay scheme requires 24 hours for enzymatic generation of a detectable product.
It has long been recognized that coupling electrochemical detection with EIA's would be advantageous. Electrodes are insensitive to the color or turbidity of a test sample, and thus can be used to develop methods directly applicable to whole blood samples. However, most of the many reports regarding the use of electrochemical detection to devise EIA's or "immunosensors" have relied on using such sensors as solid phases in heterogeneous assay arrangements in which antibodies are immobilized at the surface of a given electrode. After incubation of a sample with other reagents, the surface of the electrodes have to be washed before adding the substrate needed to measure bound enzyme activity.
As a specific example, Cozzette et al., U.S. Pat. No. 5,063,081 issued Nov. 5, 1991, discloses a ligand/ligand receptor-based biosensor for detecting a particular analyte species, such as an antigen. A base sensor comprises a catalytic indicator electrode, using a noble late transition metal, such as iridium, gold, platinum, silver and the like, is surrounded by a combined reference and counter electrode made of, for example, silver and silver chloride. (Columns 25-26.) An antibody is immobilized on the base sensor. The resulting biosensor is then brought into contact with a mixture comprising the sample and a second analyte-specific antibody, which is labeled. (Columns 45-46.) A permselective silane layer may also be used as a screen against interfering species. However, unbound materials and interfering electroactive species are preferably removed from the sensor by using either a wash solution or by using the solution containing the enzyme substrate as a wash. (Columns 47-49.)
Others using electrochemical detection methods have performed conventional heterogeneous sandwich assays by physically separating the immobilized capture antibodies, for example, in microwells or immunocolumns, from the step of electrochemically detecting the bound enzyme-antibody conjugate activity. Because the enzyme-antibody conjugate is typically present in excess, detection systems are not usually able to discriminate between the relatively small amounts of immuno-bound enzyme label and the relatively high background levels of the enzyme-labeled antibody conjugate.
Therefore, despite all of the past and current research activity in this area, there is still no single electrochemical EIA approach that enables the detection of proteins in a sample as complex as whole blood without the need to perform multiple wash steps and/or other manipulative procedures to separate the analyte and antibody materials from the substrate used to indicate the activity of a labelling enzyme. Such a non-separation EIA for proteins has long been desired.
It has now been discovered that, if the enzyme substrate is delivered only to the enzyme that is bound to the solid phase in the form of the sandwich, without simultaneously supplying substrate to the excess free enzyme-antibody conjugate in the bulk solution in any significant amount, there is no need to separate the unbound label from the bound species. Further, if the product of the enzyme reaction can be detected at the surface of a solid phase detector immediately after it is formed by spatial resolution, the analytical signal originating at the surface of the solid phase can be greatly enhanced as compared to the background signal originating from the bulk solution.